DESCRIPTION: (Scanned from the applicant's abstract) In stably transfected ER+ HeLa-ER cells, we find that 4-hydroxytamoxifen (OHT)-ER and l7b-estradiol (E2)-ER, elicit an extremely rapid activation of the p38 and Jun N-terminal kinase (JNK) pathways. Activation of p38 or iNK by OHT-ER or E2-ER is strongly correlated with the induction of apoptosis. In MCF-7 cells, OHT, but not E2, induces p38 and apoptosis and a p38 inhibitor blocks induction of p38 and apoptosis. Activation of p38 and induction of' apoptosis represent newly identified actions of estrogen receptor. The Specific Aims are: (1) To identify the pathway leading from OHT-ER (and En-ER) to the phosphorylation and activation of p38 and JNK. OHT-ER induces phosphorylation of p38 and JNK in 2 min. suggesting a nongenomic effect. (a) We will identify the ER domains (such as nuclear localization) required for OHT-ER activation of p38 and JNK; (b) evaluate the possible role in p38 activation of a membrane-associated ER using cells enriched in a putative membrane ER. by specifically targeting ER to cell membranes, and through studies of potential ER-caveolae interactions: (c) use dominant negative MKKs to determine the role of MKKs in phosphorylating p38 and JNK in response to OHT-ER and E2-ER. (2) To investigate the relationship between the activation of p38 by OHT-ER and by E2-ER and the induction of apoptosis. We will determine the kinetics of the second late stage of activation of p38 and JNK seen in our preliminary studies, determine whether apoptosis requires the late activation of p38 and JNK, and whether the late activation is genomic or non-genomic. We propose a novel, testable, hypothesis relevant to both breast cancer chemoprevention and therapy by tamoxifen (Tam), and perhaps by other SERMs. In mammary epithelial cells, the level of activation of cell death pathways produced by the DNA damage often responsible for transformation may sometimes be insufficient to induce apoptosis of all damaged cells. In cells with low levels of ER, the additional weak induction of p38 and JNK by OHT-ER potentiates apoptosis induced by DNA damage, and thereby stimulates apoptosis of pre-malignant and early stage breast cancer cells. (3) We will slightly damage the DNA of mammary epithelial cells and ER+ cell lines with a low level of UV light and determine whether Tam activates p38 or JNK and potentiates apoptosis of the cells. (4) To determine whether the progression of breast cancer cell lines to Tam resistance involves loss of Tam induction of p38 or JNK, or loss of ability of p38 or JNK to induce apoptosis, we will use existing Tam resistant breast cancer cell lines and cell lines we isolate, inhibitors and dominate negative mutants. These studies help define a new pathway for ER action and test a novel hypothesis for how SERMs function.